Adeno-associated virus (aav) serotype 8 sequences, vectors containing same, and uses therefor

ABSTRACT

Sequences of a serotype 8 adeno-associated virus and vectors and host cells containing these sequences are provided. Also described are methods of using such host cells and vectors in production of rAAV particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/598,462, filed Jan. 16, 2015, which is a divisional of U.S. patentapplication Ser. No. 11/981,022, filed Oct. 31, 2007, now U.S. Pat. No.8,962,330, issued Feb. 24, 2015, which is a continuation of U.S. patentapplication Ser. No. 11/899,500, filed Sep. 6, 2007, now U.S. Pat. No.7,790,449, issued Sep. 7, 2010, which is a continuation of U.S. patentapplication Ser. No. 10/423,704, filed Apr. 25, 2003, now U.S. Pat. No.7,282,199, issued Oct. 16, 2007, which is a continuation-in-part ofInternational Patent Application No. PCT/US02/33630, filed Nov. 12,2002, which claims the benefit under 35 USC 119(e) of U.S. ProvisionalPatent Application No. 60/386,122, filed Jun. 5, 2002, U.S. ProvisionalPatent Application No. 60/377,133, filed May 1, 2002, and U.S.Provisional Patent Application No. 60/341,151, filed Dec. 17, 2001,which applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. P30 DK47757-09 awarded by the National Institute of Diabetes and Digestive andKidney Diseases, and Grant No. P01 HL 59407-03 awarded by the NationalHeart, Lung, and Blood Institute. The government has certain rights inthe invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

The Sequence Listing material filed in electronic form herewith ishereby incorporated by reference. This file is labeled“UPN_O2733C4D1_ST25.txt”, was created on Jan. 16, 2015, and is 50,004bytes (48.8 KB).

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icosahedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus,Dependovirus, because the virus was discovered as a contaminant inpurified adenovirus stocks. AAV's life cycle includes a latent phase atwhich AAV genomes, after infection, are site specifically integratedinto host chromosomes and an infectious phase in which, following eitheradenovirus or herpes simplex virus infection, the integrated genomes aresubsequently rescued, replicated, and packaged into infectious viruses.The properties of non-pathogenicity, broad host range of infectivity,including non-dividing cells, and potential site-specific chromosomalintegration make AAV an attractive tool for gene transfer.

Recent studies suggest that AAV vectors may be the preferred vehicle forgene delivery. To date, there have been 6 different serotypes of AAVsisolated from human or non-human primates (NHP) and well characterized.Among them, human serotype 2 is the first AAV that was developed as agene transfer vector; it has been widely used for efficient genetransfer experiments in different target tissues and animal models.Clinical trials of the experimental application of AAV2 based vectors tosome human disease models are in progress, and include such diseases ascystic fibrosis and hemophilia B.

What are desirable are AAV-based constructs for gene delivery.

SUMMARY OF THE INVENTION

In one aspect, the invention provides novel AAV sequences, compositionscontaining these sequences, and uses therefor. Advantageously, thesecompositions are particularly well suited for use in compositionsrequiring re-administration of rAAV for therapeutic or prophylacticpurposes.

These and other aspects of the invention will be readily apparent fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C are the nucleic acid sequences of the rep and capregions of AAV8 [SEQ ID NO:1].

FIGS. 2A through 2C are the amino acid sequences of the AAV8 capsid vp1protein [SEQ ID NO:2], provided in alignment with the vp1 of thepublished sequences of AAV2 [SEQ ID NO:4], AAV1 [SEQ ID NO:5], and AAV3[SEQ ID NO:6], and newly identified AAV serotypes AAV7 [SEQ ID NO: 8]and AAV9 [SEQ ID NO:7]. The alignment was performed using the Clustal Wprogram, with the number of AAV2 used for reference. Underlining andbold at the bottom sequence of the alignment indicates cassettes ofidentity. The dots in the alignment indicate that the amino acids aremissing at the positions in the alignment as compared to AAV2 VP1.

FIGS. 3A through 3C are the amino acid sequences of the AAV8 repproteins [SEQ ID NO:3].

DETAILED DESCRIPTION OF THE INVENTION

The invention provides the nucleic acid sequences and amino acids of anovel AAV serotype, AAV8. Also provided are fragments of these AAVsequences. Each of these fragments may be readily utilized in a varietyof vector systems and host cells. Among desirable AAV8 fragments are thecap proteins, including the vp1, vp2, vp3 and hypervariable regions, therep proteins, including rep 78, rep 68, rep 52, and rep 40, and thesequences encoding these proteins. These fragments may be readilyutilized in a variety of vector systems and host cells. Such fragmentsmay be used alone, in combination with other AAV8 sequences orfragments, or in combination with elements from other AAV or non-AAVviral sequences. In one particularly desirable embodiment, a vectorcontains the AAV8 cap and/or rep sequences of the invention.

The AAV8 sequences and fragments thereof are useful in production ofrAAV, and are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. The invention further provides nucleic acidmolecules, gene delivery vectors, and host cells which contain the AAV8sequences of the invention.

Suitable fragments can be determined using the information providedherein. Alignments are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs, such as“Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similar programs are available for amino acidsequences, e.g., the “Clustal X” program. Generally, any of theseprograms are used at default settings, although one of skill in the artcan alter these settings as needed. Alternatively, one of skill in theart can utilize another algorithm or computer program which provides atleast the level of identity or alignment as that provided by thereferenced algorithms and programs.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or anopen reading frame thereof, or another suitable fragment which is atleast 15 nucleotides in length. Examples of suitable fragments aredescribed herein.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, e.g., a cap protein, a rep protein, or a fragmentthereof which is at least 8 amino acids, or more desirably, at least 15amino acids in length. Examples of suitable fragments are describedherein.

By the term “highly conserved” is meant at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. Identity is readily determined by one of skill in the art byresort to algorithms and computer programs known by those of skill inthe art.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences whichare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome,the full-length of a gene coding sequence, or a fragment of at leastabout 500 to 5000 nucleotides, is desired. However, identity amongsmaller fragments, e.g. of at least about nine nucleotides, usually atleast about 20 to 24 nucleotides, at least about 28 to 32 nucleotides,at least about 36 or more nucleotides, may also be desired. Similarly,“percent sequence identity” may be readily determined for amino acidsequences, over the full-length of a protein, or a fragment thereof.Suitably, a fragment is at least about 8 amino acids in length, and maybe up to about 700 amino acids. Examples of suitable fragments aredescribed herein.

As described herein, the vectors of the invention containing the AAVcapsid proteins of the invention are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other AAV serotype based vectors, as well as otherviral vectors. The rAAV vectors of the invention are particularlyadvantageous in rAAV readministration and repeat gene therapy.

These and other embodiments and advantages of the invention aredescribed in more detail below. As used throughout this specificationand the claims, the terms “comprising” and “including” are inclusive ofother components, elements, integers, steps and the like. Conversely,the term “consisting” and its variants are exclusive of othercomponents, elements, integers, steps and the like.

I. AAV Serotype 8 Sequences

A. Nucleic Acid Sequences

The AAV8 nucleic acid sequences of the invention include the DNAsequences of FIG. 1 [SEQ ID NO: 1], which consists of 4396 nucleotides.The AAV8 nucleic acid sequences of the invention further encompass thestrand which is complementary to FIG. 1 [SEQ ID NO: 1], as well as theRNA and cDNA sequences corresponding to FIG. 1 [SEQ ID NO: 1] and itscomplementary strand. Also included in the nucleic acid sequences of theinvention are natural variants and engineered modifications of FIG. 1[SEQ ID NO: 1] and its complementary strand. Such modifications include,for example, labels which are known in the art, methylation, andsubstitution of one or more of the naturally occurring nucleotides witha degenerate nucleotide.

Further included in this invention are nucleic acid sequences which aregreater than about 90%, more preferably at least about 95%, and mostpreferably at least about 98 to 99% identical or homologous to FIG. 1[SEQ ID NO:1].

Also included within the invention are fragments of FIG. 1 [SEQ ID NO:1], its complementary strand, cDNA and RNA complementary thereto.Suitable fragments are at least 15 nucleotides in length, and encompassfunctional fragments, i.e., fragments which are of biological interest.Such fragments include the sequences encoding the three variableproteins (vp) of the AAV8 capsid which are alternative splice variants:vp1 [nt 2121 to 4335 of FIG. 1, SEQ ID NO:1]; vp2 [nt 2532 to 4335 ofFIG. 1, SEQ ID NO:1]; and vp 3 [nt 2730 to 4335 of FIG. 1, SEQ ID NO:1].Other suitable fragments of FIG. 1 [SEQ ID NO:1], include the fragmentwhich contains the start codon for the AAV8 capsid protein, and thefragments encoding the hypervariable regions of the vp1 capsid protein,which are described herein.

Still other fragments include those encoding the rep proteins, includingrep 78 [initiation codon located at nt 227 of FIG. 1, SEQ ID NO:1], rep68 [initiation codon located at nt 227 of FIG. 1, SEQ ID NO:1], rep 52[initiation codon located at nt 905 of FIG. 1, SEQ ID NO:1], and rep 40[initiation codon located at nt 905 of FIG. 1, SEQ ID NO:1]. Otherfragments of interest may include the AAV8 inverted terminal repeatwhich can be identified by the methods described herein, AAV P19sequences, AAV8 P40 sequences, the rep binding site, and the terminalresolute site (TRS). Still other suitable fragments will be readilyapparent to those of skill in the art.

In addition to including the nucleic acid sequences provided in thefigures and Sequence Listing, the present invention includes nucleicacid molecules and sequences which are designed to express the aminoacid sequences, proteins and peptides of the AAV serotypes of theinvention. Thus, the invention includes nucleic acid sequences whichencode the following novel AAV amino acid sequences and artificial AAVserotypes generated using these sequences and/or unique fragmentsthereof.

As used herein, artificial AAV serotypes include, without limitation,AAV with a non-naturally occurring capsid protein. Such an artificialcapsid may be generated by any suitable technique, using a novel AAVsequence of the invention (e.g., a fragment of a vp1 capsid protein) incombination with heterologous sequences which may be obtained fromanother AAV serotype (known or novel), non-contiguous portions of thesame AAV serotype, from a non-AAV viral source, or from a non-viralsource. An artificial AAV serotype may be, without limitation, achimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAVcapsid.

B. AAV8 Amino Acid Sequences, Proteins and Peptides

The invention further provides proteins and fragments thereof which areencoded by the AAV8 nucleic acids of the invention, and AAV8 amino acidswhich are generated by other methods. The invention further encompassesAAV serotypes generated using sequences of the novel AAV serotype of theinvention, which are generated using synthetic, recombinant or othertechniques known to those of skill in the art. The invention is notlimited to novel AAV amino acid sequences, peptides and proteinsexpressed from the novel AAV nucleic acid sequences of the invention andencompasses amino acid sequences, peptides and proteins generated byother methods known in the art, including, e.g., by chemical synthesis,by other synthetic techniques, or by other methods. For example, thesequences of any of be readily generated using a variety of techniques.

Suitable production techniques are well known to those of skill in theart. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively,peptides can also be synthesized by the well known solid phase peptidesynthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962);Stewart and Young, Solid Phase Peptide Synthesis (Freeman, SanFrancisco, 1969) pp. 27-62). These and other suitable production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

Particularly desirable proteins include the AAV capsid proteins, whichare encoded by the nucleotide sequences identified above. The AAV capsidis composed of three proteins, vp1, vp2 and vp3, which are alternativesplice variants. The full-length sequence provided in FIG. 2 is that ofvp1. The AAV8 capsid proteins include vp1 [aa 1 to 737 of SEQ ID NO:2],vp2 [aa 138 to 737 of SEQ ID NO:2], and vp3 [aa 203 to 737 of SEQ ID NO:2] and functional fragments thereof. Other desirable fragments of thecapsid protein include the constant and variable regions, locatedbetween hypervariable regions (HPV). Other desirable fragments of thecapsid protein include the HPV themselves.

An algorithm developed to determine areas of sequence divergence in AAV2has yielded 12 hypervariable regions (HVR) of which 5 overlap or arepart of the four previously described variable regions. [Chiorini et al,J. Virol, 73:1309-19 (1999); Rutledge et al, J. Virol., 72:309-319]Using this algorithm and/or the alignment techniques described herein,the HVR of the novel AAV serotypes are determined. For example, withrespect to the number of the AAV2 vp1 [SEQ ID NO:4], the HVR are locatedas follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa 262-264; HVR4,aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa500-504; HVR8,aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594; HVR11, aa 658-667; andHVR12, aa 705-719. Using the alignment provided herein performed usingthe Clustal X program at default settings, or using other commerciallyor publicly available alignment programs at default settings, one ofskill in the art can readily determine corresponding fragments of thenovel AAV capsids of the invention.

Still other desirable fragments of the AAV8 capsid protein include aminoacids 1 to 184 of SEQ ID NO: 2, amino acids 199 to 259; amino acids 274to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids 724to 736 of SEQ ID NO:2; aa 185-198; aa 260-273; aa447-477; aa495-602;aa660-669; and aa707-723. Additionally, examples of other suitablefragments of AAV capsids include, with respect to the numbering of AAV2[SEQ ID NO:4], aa 24-42, aa 25-28; aa 81-85; aa133-165; aa 134-165; aa137-143; aa 154-156; aa 194-208; aa 261-274; aa 262-274; aa 171-173; aa413-417; aa 449-478; aa 494-525; aa 534-571; aa 581-601; aa 660-671; aa709-723. Still other desirable fragments include, for example, in AAV7,amino acids 1 to 184 of SEQ ID NO:2, amino acids 199 to 259; amino acids274 to 446; amino acids 603 to 659; amino acids 670 to 706; amino acids724 to 736; aa 185 to 198; aa 260 to 273; aa447 to 477; aa495 to 602;aa660 to 669; and aa707 to 723. Using the alignment provided hereinperformed using the Clustal X program at default settings, or usingother commercially or publicly available alignment programs at defaultsettings, one of skill in the art can readily determine correspondingfragments of the novel AAV capsids of the invention.

Still other desirable AAV8 proteins include the rep proteins includerep68/78 and rep40/52 [located within aa 1 to 625 of SEQ ID NO: 3].Suitable fragments of the rep proteins may include aa 1 to 102; aa 103to 140; aa 141 to 173; aa 174 to 226; aa 227 to 275; aa 276 to 374; aa375 to 383; aa 384 to 446; aa 447 to 542; aa 543 to 555; aa 556 to 625,of SEQ ID NO: 3.

Suitably, fragments are at least 8 amino acids in length. However,fragments of other desired lengths may be readily utilized. Suchfragments may be produced recombinantly or by other suitable means,e.g., chemical synthesis.

The invention further provides other AAV8 sequences which are identifiedusing the sequence information provided herein. For example, given theAAV8 sequences provided herein, infectious AAV8 may be isolated usinggenome walking technology (Siebert et al., 1995, Nucleic Acid Research,23:1087-1088, Friezner-Degen et al., 1986, J. Biol. Chem. 261:6972-6985,BD Biosciences Clontech, Palo Alto, Calif.). Genome walking isparticularly well suited for identifying and isolating the sequencesadjacent to the novel sequences identified according to the method ofthe invention. This technique is also useful for isolating invertedterminal repeat (ITRs) of the novel AAV8 serotype, based upon the novelAAV capsid and rep sequences provided herein.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Such production methods are withinthe knowledge of those of skill in the art and are not a limitation ofthe present invention.

IV. Production of rAAV with AAV8 Capsids

The invention encompasses novel, wild-type AAV8, the sequences of whichare free of DNA and/or cellular material with these viruses areassociated in nature. In another aspect, the present invention providesmolecules which utilize the novel AAV sequences of the invention,including fragments thereof, for production of molecules useful indelivery of a heterologous gene or other nucleic acid sequences to atarget cell.

In another aspect, the present invention provides molecules whichutilize the AAV8 sequences of the invention, including fragmentsthereof, for production of viral vectors useful in delivery of aheterologous gene or other nucleic acid sequences to a target cell.

The molecules of the invention which contain AAV8 sequences include anygenetic element (vector) which may be delivered to a host cell, e.g.,naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in anon-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc.which transfer the sequences carried thereon. The selected vector may bedelivered by any suitable method, including transfection,electroporation, liposome delivery, membrane fusion techniques, highvelocity DNA-coated pellets, viral infection and protoplast fusion. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.

In one embodiment, the vectors of the invention contain, at a minimum,sequences encoding an AAV8 capsid or a fragment thereof. In anotherembodiment, the vectors of the invention contain, at a minimum,sequences encoding an AAV8 rep protein or a fragment thereof.Optionally, such vectors may contain both AAV cap and rep proteins. Invectors in which both AAV rep and cap are provides, the AAV rep and AAVcap sequences can both be of AAV8 origin. Alternatively, the presentinvention provides vectors in which the rep sequences are from an AAVserotype which differs from that which is providing the cap sequences.In one embodiment, the rep and cap sequences are expressed from separatesources (e.g., separate vectors, or a host cell and a vector). Inanother embodiment, these rep sequences are fused in frame to capsequences of a different AAV serotype to form a chimeric AAV vector.Optionally, the vectors of the invention further contain a minigenecomprising a selected transgene which is flanked by AAV 5′ ITR and AAV3′ ITR.

Thus, in one embodiment, the vectors described herein contain nucleicacid sequences encoding an intact AAV capsid which may be from a singleAAV serotype (e.g., AAV8). Such a capsid may comprise amino acids 1 to738 of SEQ ID NO:2. Alternatively, these vectors contain sequencesencoding artificial capsids which contain one or more fragments of theAAV8 capsid fused to heterologous AAV or non-AAV capsid proteins (orfragments thereof). These artificial capsid proteins are selected fromnon-contiguous portions of the AAV8 capsid or from capsids of other AAVserotypes. For example, a rAAV may have a capsid protein comprising oneor more of the AAV8 capsid regions selected from the vp2 and/or vp3, orfrom vp 1, or fragments thereof selected from amino acids 1 to 184,amino acids 199 to 259; amino acids 274 to 446; amino acids 603 to 659;amino acids 670 to 706; amino acids 724 to 738 of the AAV8 capsid, SEQID NO: 2. In another example, it may be desirable to alter the startcodon of the vp3 protein to GTG. Alternatively, the rAAV may contain oneor more of the AAV serotype 8 capsid protein hypervariable regions whichare identified herein, or other fragment including, without limitation,aa 185-198; aa 260-273; aa447-477; aa495-602; aa660-669; and aa707-723of the AAV8 capsid. See, SEQ ID NO: 2. These modifications may be toincrease expression, yield, and/or to improve purification in theselected expression systems, or for another desired purpose (e.g., tochange tropism or alter neutralizing antibody epitopes).

The vectors described herein, e.g., a plasmid, are useful for a varietyof purposes, but are particularly well suited for use in production of arAAV containing a capsid comprising AAV sequences or a fragment thereof.These vectors, including rAAV, their elements, construction, and usesare described in detail herein.

In one aspect, the invention provides a method of generating arecombinant adeno-associated virus (AAV) having an AAV serotype 8capsid, or a portion thereof. Such a method involves culturing a hostcell which contains a nucleic acid sequence encoding an adeno-associatedvirus (AAV) serotype 8 capsid protein, or fragment thereof, as definedherein; a functional rep gene; a minigene composed of, at a minimum, AAVinverted terminal repeats (ITRs) and a transgene; and sufficient helperfunctions to permit packaging of the minigene into the AAV8 capsidprotein.

The components required to be cultured in the host cell to package anAAV minigene in an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,minigene, rep sequences, cap sequences, and/or helper functions) may beprovided by a stable host cell which has been engineered to contain oneor more of the required components using methods known to those of skillin the art. Most suitably, such a stable host cell will contain therequired component(s) under the control of an inducible promoter.However, the required component(s) may be under the control of aconstitutive promoter. Examples of suitable inducible and constitutivepromoters are provided herein, in the discussion of regulatory elementssuitable for use with the transgene. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated which is derived from 293 cells (whichcontain E1 helper functions under the control of a constitutivepromoter), but which contains the rep and/or cap proteins under thecontrol of inducible promoters. Still other stable host cells may begenerated by one of skill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transferthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

Unless otherwise specified, the AAV ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAVserotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV9 and the novel serotype of the invention, AAV8. TheseITRs or other AAV components may be readily isolated using techniquesavailable to those of skill in the art from an AAV serotype. Such AAVmay be isolated or obtained from academic, commercial, or public sources(e.g., the American Type Culture Collection, Manassas, Va.).Alternatively, the AAV sequences may be obtained through synthetic orother suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank,PubMed, or the like.

A. The Minigene

The minigene is composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). In one desirable embodiment, the ITRs of AAV serotype 2 areused. However, ITRs from other suitable serotypes may be selected. It isthis minigene which is packaged into a capsid protein and delivered to aselected host cell.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc.

These coding sequences, when associated with regulatory elements whichdrive their expression, provide signals detectable by conventionalmeans, including enzymatic, radiographic, colorimetric, fluorescence orother spectrographic assays, fluorescent activating cell sorting assaysand immunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for beta-galactosidaseactivity. Where the transgene is green fluorescent protein orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

However, desirably, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs.Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalyticRNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs.One example of a useful RNA sequence is a sequence which inhibits orextinguishes expression of a targeted nucleic acid sequence in thetreated animal. Typically, suitable target sequences in includeoncologic targets and viral diseases. See, for examples of such targetsthe oncologic targets and viruses identified below in the sectionrelating to immunogens.

The transgene may be used to correct or ameliorate gene deficiencies,which may include deficiencies in which normal genes are expressed atless than normal levels or deficiencies in which the functional geneproduct is not expressed. A preferred type of transgene sequence encodesa therapeutic protein or polypeptide which is expressed in a host cell.The invention further includes using multiple transgenes, e.g., tocorrect or ameliorate a gene defect caused by a multi-subunit protein.In certain situations, a different transgene may be used to encode eachsubunit of a protein, or to encode different peptides or proteins. Thisis desirable when the size of the DNA encoding the protein subunit islarge, e.g., for an immunoglobulin, the platelet-derived growth factor,or a dystrophin protein. In order for the cell to produce themulti-subunit protein, a cell is infected with the recombinant viruscontaining each of the different subunits. Alternatively, differentsubunits of a protein may be encoded by the same transgene. In thiscase, a single transgene includes the DNA encoding each of the subunits,with the DNA for each subunit separated by an internal ribozyme entrysite (IRES). This is desirable when the size of the DNA encoding each ofthe subunits is small, e.g., the total size of the DNA encoding thesubunits and the IRES is less than five kilobases. As an alternative toan IRES, the DNA may be separated by sequences encoding a 2A peptide,which self-cleaves in a post-translational event. See, e.g., M. L.Donnelly, et al, J. Gen. Virol., 78(Pt 1):13-21 (January 1997); Furler,S., et al, Gene Ther., 8(11):864-873 (June 2001); Klump H., et al., GeneTher., 8(10):811-817 (May 2001). This 2A peptide is significantlysmaller than an IRES, making it well suited for use when space is alimiting factor. More often, when the transgene is large, consists ofmulti-subunits, or two transgenes are co-delivered, rAAV carrying thedesired transgene(s) or subunits are co-administered to allow them toconcatamerize in vivo to form a single vector genome. In such anembodiment, a first AAV may carry an expression cassette which expressesa single transgene and a second AAV may carry an expression cassettewhich expresses a different transgene for co-expression in the hostcell. However, the selected transgene may encode any biologically activeproduct or other product, e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis invention.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements which are operablylinked to the transgene in a manner which permits its transcription,translation and/or expression in a cell transfected with the plasmidvector or infected with the virus produced by the invention. As usedherein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1promoter [Invitrogen]. Inducible promoters allow regulation of geneexpression and can be regulated by exogenously supplied compounds,environmental factors such as temperature, or the presence of a specificphysiological state, e.g., acute phase, a particular differentiationstate of the cell, or in replicating cells only. Inducible promoters andinducible systems are available from a variety of commercial sources,including, without limitation, Invitrogen, Clontech and Ariad. Manyother systems have been described and can be readily selected by one ofskill in the art. Examples of inducible promoters regulated byexogenously supplied compounds, include, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system[WO 98/10088]; the ecdysone insect promoter [No et al, Proc. Natl. Acad.Sci. USA, 93:3346-3351 (1996)], the tetracycline-repressible system[Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)], thetetracycline-inducible system [Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)], the RU486-inducible system [Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)] and therapamycin-inducible system [Magari et al, J. Clin. Invest.,100:2865-2872 (1997)]. Other types of inducible promoters which may beuseful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particulardifferentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a gene operably linked to atissue-specific promoter. For instance, if expression in skeletal muscleis desired, a promoter active in muscle should be used. These includethe promoters from genes encoding skeletal β-actin, myosin light chain2A, dystrophin, muscle creatine kinase, as well as synthetic musclepromoters with activities higher than naturally-occurring promoters (seeLi et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters thatare tissue-specific are known for liver (albumin, Miyatake et al., J.Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig etal., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot etal., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al.,Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptorchain), neuronal such as neuron-specific enolase (NSE) promoter(Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)),neurofilament light-chain gene (Piccioli et al., Proc. Natl. Acad. Sci.USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli etal., Neuron, 15:373-84 (1995)), among others.

Optionally, plasmids carrying therapeutically useful transgenes may alsoinclude selectable markers or reporter genes may include sequencesencoding geneticin, hygromicin or purimycin resistance, among others.Such selectable reporters or marker genes (preferably located outsidethe viral genome to be rescued by the method of the invention) can beused to signal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the plasmid may include anorigin of replication. Selection of these and other promoters and vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The combination of the transgene, promoter/enhancer, and 5′ and 3′ ITRsis referred to as a “minigene” for ease of reference herein. Providedwith the teachings of this invention, the design of such a minigene canbe made by resort to conventional techniques.

3. Delivery of the Minigene to a Packaging Host Cell

The minigene can be carried on any suitable vector, e.g., a plasmid,which is delivered to a host cell. The plasmids useful in this inventionmay be engineered such that they are suitable for replication and,optionally, integration in prokaryotic cells, mammalian cells, or both.These plasmids (or other vectors carrying the 5′ AAV ITR-heterologousmolecule-3′ITR) contain sequences permitting replication of the minigenein eukaryotes and/or prokaryotes and selection markers for thesesystems. Selectable markers or reporter genes may include sequencesencoding geneticin, hygromicin or purimycin resistance, among others.The plasmids may also contain certain selectable reporters or markergenes that can be used to signal the presence of the vector in bacterialcells, such as ampicillin resistance. Other components of the plasmidmay include an origin of replication and an amplicon, such as theamplicon system employing the Epstein Barr virus nuclear antigen. Thisamplicon system, or other similar amplicon components permit high copyepisomal replication in the cells. Preferably, the molecule carrying theminigene is transfected into the cell, where it may exist transiently.Alternatively, the minigene (carrying the 5′ AAV ITR-heterologousmolecule-3′ ITR) may be stably integrated into the genome of the hostcell, either chromosomally or as an episome. In certain embodiments, theminigene may be present in multiple copies, optionally in head-to-head,head-to-tail, or tail-to-tail concatamers. Suitable transfectiontechniques are known and may readily be utilized to deliver the minigeneto the host cell.

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, about 10 to about 50 μg DNA to about 1×10⁴ cells toabout 1×10¹³ cells, or about 10⁵ cells. However, the relative amounts ofvector DNA to host cells may be adjusted, taking into consideration suchfactors as the selected vector, the delivery method and the host cellsselected.

B. Rep and Cap Sequences

In addition to the minigene, the host cell contains the sequences whichdrive expression of the AAV8 capsid protein (or a capsid proteincomprising a fragment of the AAV8 capsid) in the host cell and repsequences of the same serotype as the serotype of the AAV ITRs found inthe minigene, or a cross-complementing serotype. The AAV cap and repsequences may be independently obtained from an AAV source as describedabove and may be introduced into the host cell in any manner known toone in the art as described above. Additionally, when pseudotyping anAAV vector in an AAV8 capsid, the sequences encoding each of theessential rep proteins may be supplied by AAV8, or the sequencesencoding the rep proteins may be supplied by different AAV serotypes(e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9). For example, therep78/68 sequences may be from AAV2, whereas the rep52/40 sequences maybe from AAV8.

In one embodiment, the host cell stably contains the capsid proteinunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the capsid protein is expressedunder the control of an inducible promoter. In another embodiment, thecapsid protein is supplied to the host cell in trans. When delivered tothe host cell in trans, the capsid protein may be delivered via aplasmid which contains the sequences necessary to direct expression ofthe selected capsid protein in the host cell. Most desirably, whendelivered to the host cell in trans, the plasmid carrying the capsidprotein also carries other sequences required for packaging the rAAV,e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell. Mostdesirably, when delivered to the host cell in trans, the plasmidcarrying the capsid protein also carries other sequences required forpackaging the rAAV, e.g., the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the chromosome of the cell. Anotherembodiment has the rep and cap sequences transiently expressed in thehost cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the minigene. The vector may comprise one or more of thegenes encoding the helper functions, e.g., the adenoviral proteins E1,E2a, and E4ORF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In one embodiment, an AAV P5 promotersequence is employed. The selection of the AAV to provide any of thesesequences does not limit the invention.

In another preferred embodiment, the promoter for rep is an induciblepromoter, such as are discussed above in connection with the transgeneregulatory elements. One preferred promoter for rep expression is the T7promoter. The vector comprising the rep gene regulated by the T7promoter and the cap gene, is transfected or transformed into a cellwhich either constitutively or inducibly expresses the T7 polymerase.See WO 98/10088, published Mar. 12, 1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the phage laddersequences or yeast ladder sequences, which are available commercially,e.g., from Gibco or Invitrogen, among others. The spacer may be of anysize sufficient to reduce expression of the rep78 and rep68 geneproducts, leaving the rep52, rep40 and cap gene products expressed atnormal levels. The length of the spacer may therefore range from about10 bp to about 10.0 kbp, preferably in the range of about 100 bp toabout 8.0 kbp. To reduce the possibility of recombination, the spacer ispreferably less than 2 kbp in length; however, the invention is not solimited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions may besupplied by a herpesvirus. Most desirably, the necessary helperfunctions are each provided from a human or non-human primate adenovirussource, such as those described above and/or are available from avariety of sources, including the American Type Culture Collection(ATCC), Manassas, Va. (US). In one currently preferred embodiment, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Thehost cell may contain other adenoviral genes such as VAI RNA, but thesegenes are not required. In a preferred embodiment, no other adenovirusgenes or gene functions are present in the host cell.

By “adenoviral DNA which expresses the E1a gene product”, it is meantany adenovirus sequence encoding E1a or any functional E1a portion.Adenoviral DNA which expresses the E2a gene product and adenoviral DNAwhich expresses the E4 ORF6 gene products are defined similarly. Alsoincluded are any alleles or other modifications of the adenoviral geneor functional portion thereof. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the adenoviral function in some manner, as well asnaturally occurring allelic variants thereof. Such modifications andmethods for manipulating DNA to achieve these adenovirus gene functionsare known to those of skill in the art.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by exogenously added factors, forexample.

D. Host Cells and Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for thecell used is that it not carry any adenovirus gene other than E1, E2aand/or E4 ORF6; it not contain any other virus gene which could resultin homologous recombination of a contaminating virus during theproduction of rAAV; and it is capable of infection or transfection ofDNA and expression of the transfected DNA. In a preferred embodiment,the host cell is one that has rep and cap stably transfected in thecell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the minigene as described above. Stable rep and/or capexpressing cell lines, such as B-50 (PCT/US98/19463), or those describedin U.S. Pat. No. 5,658,785, may also be similarly employed. Anotherdesirable host cell contains the minimum adenoviral DNA which issufficient to express E4 ORF6. Yet other cell lines can be constructedusing the AAV8 rep and/or AAV8 cap sequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

The AAV8 based vectors which are generated by one of skill in the artare beneficial for gene delivery to selected host cells and gene therapypatients since no neutralization antibodies to AAV8 have been found inthe human population. One of skill in the art may readily prepare otherrAAV viral vectors containing the AAV8 capsid proteins provided hereinusing a variety of techniques known to those of skill in the art. Onemay similarly prepare still other rAAV viral vectors containing AAV8sequence and AAV capsids of another serotype.

One of skill in the art will readily understand that the AAV8 sequencesof the invention can be readily adapted for use in these and other viralvector systems for in vitro, ex vivo or in vivo gene delivery.Similarly, one of skill in the art can readily select other fragments ofthe AAV8 genome of the invention for use in a variety of rAAV andnon-rAAV vector systems. Such vectors systems may include, e.g.,lentiviruses, retroviruses, poxviruses, vaccinia viruses, and adenoviralsystems, among others. Selection of these vector systems is not alimitation of the present invention.

Thus, the invention further provides vectors generated using the nucleicacid and amino acid sequences of the novel AAV of the invention. Suchvectors are useful for a variety of purposes, including for delivery oftherapeutic molecules and for use in vaccine regimens. Particularlydesirable for delivery of therapeutic molecules are recombinant AAVcontaining capsids of the novel AAV of the invention. These, or othervector constructs containing novel AAV sequences of the invention may beused in vaccine regimens, e.g., for co-delivery of a cytokine, or fordelivery of the immunogen itself.

V. Recombinant Viruses and Uses Therefor

Using the techniques described herein, one of skill in the art cangenerate a rAAV having a capsid of a serotype 8 of the invention orhaving a capsid containing one or more fragments of AAV8. In oneembodiment, a full-length capsid from a single serotype, e.g., AAV8 [SEQID NO: 2] can be utilized. In another embodiment, a full-length capsidmay be generated which contains one or more fragments of AAV8 fused inframe with sequences from another selected AAV serotype, or fromheterologous portions of AAV8. For example, a rAAV may contain one ormore of the novel hypervariable region sequences of AAV8. Alternatively,the unique AAV8 sequences of the invention may be used in constructscontaining other viral or non-viral sequences. Optionally, a recombinantvirus may carry AAV8 rep sequences encoding one or more of the AAV8 repproteins.

A. Delivery of Viruses

In another aspect, the present invention provides a method for deliveryof a transgene to a host which involves transfecting or infecting aselected host cell with a recombinant viral vector generated with theAAV8 sequences (or functional fragments thereof) of the invention.Methods for delivery are well known to those of skill in the art and arenot a limitation of the present invention.

In one desirable embodiment, the invention provides a method for AAV8mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequenceswhich direct expression thereof and AAV8 capsid proteins.

Optionally, a sample from the host may be first assayed for the presenceof antibodies to a selected AAV serotype. A variety of assay formats fordetecting neutralizing antibodies are well known to those of skill inthe art. The selection of such an assay is not a limitation of thepresent invention. See, e.g., Fisher et al, Nature Med., 3(3):306-312(March 1997) and W C Manning et al, Human Gene Therapy, 9:477-485 (Mar.1, 1998). The results of this assay may be used to determine which AAVvector containing capsid proteins of a particular serotype are preferredfor delivery, e.g., by the absence of neutralizing antibodies specificfor that capsid serotype.

In one aspect of this method, the delivery of vector with AAV8 capsidproteins may precede or follow delivery of a gene via a vector with adifferent serotype AAV capsid protein. Thus, gene delivery via rAAVvectors may be used for repeat gene delivery to a selected host cell.Desirably, subsequently administered rAAV vectors carry the sametransgene as the first rAAV vector, but the subsequently administeredvectors contain capsid proteins of serotypes which differ from the firstvector. For example, if a first vector has AAV8 capsid proteins,subsequently administered vectors may have capsid proteins selected fromamong the other serotypes.

Optionally, multiple rAAV8 vectors can be used to deliver largetransgenes or multiple transgenes by co-administration of rAAV vectorsconcatamerize in vivo to form a single vector genome. In such anembodiment, a first AAV may carry an expression cassette which expressesa single transgene (or a subunit thereof) and a second AAV may carry anexpression cassette which expresses a second transgene (or a differentsubunit) for co-expression in the host cell. A first AAV may carry anexpression cassette which is a first piece of a polycistronic construct(e.g., a promoter and transgene, or subunit) and a second AAV may carryan expression cassette which is a second piece of a polycistronicconstruct (e.g., transgene or subunit and a polyA sequence). These twopieces of a polycistronic construct concatamerize in vivo to form asingle vector genome which co-expresses the transgenes delivered by thefirst and second AAV. In such embodiments, the rAAV vector carrying thefirst expression cassette and the rAAV vector carrying the secondexpression cassette can be delivered in a single pharmaceuticalcomposition. In other embodiments, the two or more rAAV vectors aredelivered as separate pharmaceutical compositions which can beadministered substantially simultaneously, or shortly before or afterone another.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian patient. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The vectors are administered in sufficient amounts to transfect thecells and to provide sufficient levels of gene transfer and expressionto provide a therapeutic benefit without undue adverse effects, or withmedically acceptable physiological effects, which can be determined bythose skilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include, but are not limited to,direct delivery to a desired organ (e.g., the liver (optionally via thehepatic artery) or lung), oral, inhalation, intranasal, intratracheal,intraarterial, intraocular, intravenous, intramuscular, subcutaneous,intradermal, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about0.1 ml to about 100 ml of solution containing concentrations of fromabout 1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage fordelivery to large organs (e.g., liver, muscle, heart and lung) may beabout 5×10¹⁰ to 5×10¹³ AAV genomes per 1 kg, at a volume of about 1 to100 mL. A preferred dosage for delivery to eye is about 5×10⁹ to 5×10¹²genome copies, at a volume of about 0.1 mL to 1 mL. The dosage will beadjusted to balance the therapeutic benefit against any side effects andsuch dosages may vary depending upon the therapeutic application forwhich the recombinant vector is employed. The levels of expression ofthe transgene can be monitored to determine the frequency of dosageresulting in viral vectors, preferably AAV vectors containing theminigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention.

Examples of therapeutic products and immunogenic products for deliveryby the AAV8-containing vectors of the invention are provided below.These vectors may be used for a variety of therapeutic or vaccinalregimens, as described herein. Additionally, these vectors may bedelivered in combination with one or more other vectors or activeingredients in a desired therapeutic and/or vaccinal regimen.

B. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), platelet-derived growth factor (PDGF), insulin growth factors Iand II (IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregluin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors α and β,interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses gene products suchas members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence. Still other useful gene products includeenzymes such as may be useful in enzyme replacement therapy, which isuseful in a variety of conditions resulting from deficient activity ofenzyme. For example, enzymes that contain mannose-6-phosphate may beutilized in therapies for lysosomal storage diseases (e.g., a suitablegene includes that encoding β-glucuronidase (GUSB)).

Still other useful gene products include those used for treatment ofhemophilia, including hemophilia B (including Factor IX) and hemophiliaA (including Factor VIII and its variants, such as the light chain andheavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No.6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for2351 amino acids and the protein has six domains, designated from theamino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al,Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Tooleet al, Nature, 342:337 (1984)]. Human Factor VIII is processed withinthe cell to yield a heterodimer primarily comprising a heavy chaincontaining the A1, A2 and B domains and a light chain containing the A3,C1 and C2 domains. Both the single chain polypeptide and the heterodimercirculate in the plasma as inactive precursors, until activated bythrombin cleavage between the A2 and B domains, which releases the Bdomain and results in a heavy chain consisting of the A1 and A2 domains.The B domain is deleted in the activated procoagulant form of theprotein. Additionally, in the native protein, two polypeptide chains(“a” and “b”), flanking the B domain, are bound to a divalent calciumcation.

In some embodiments, the minigene comprises first 57 base pairs of theFactor VIII heavy chain which encodes the 10 amino acid signal sequence,as well as the human growth hormone (hGH) polyadenylation sequence. Inalternative embodiments, the minigene further comprises the A1 and A2domains, as well as 5 amino acids from the N-terminus of the B domain,and/or 85 amino acids of the C-terminus of the B domain, as well as theA3, C1 and C2 domains. In yet other embodiments, the nucleic acidsencoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount of AAVvector that produces sufficient amounts of Factor VIII to decrease thetime it takes for a subject's blood to clot. Generally, severehemophiliacs having less than 1% of normal levels of Factor VIII have awhole blood clotting time of greater than 60 minutes as compared toapproximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIIIsequence. Many natural and recombinant forms of Factor VIII have beenisolated and generated. Examples of naturally occurring and recombinantforms of Factor VII can be found in the patent and scientific literatureincluding, U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521, U.S. Pat.No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No. 4,757,006, U.S.Pat. No. 5,661,008, U.S. Pat. No. 5,789,203, U.S. Pat. No. 5,681,746,U.S. Pat. No. 5,595,886, U.S. Pat. No. 5,045,455, U.S. Pat. No.5,668,108, U.S. Pat. No. 5,633,150, U.S. Pat. No. 5,693,499, U.S. Pat.No. 5,587,310, U.S. Pat. No. 5,171,844, U.S. Pat. No. 5,149,637, U.S.Pat. No. 5,112,950, U.S. Pat. No. 4,886,876, WO 94/11503, WO 87/07144,WO 92/16557, WO 91/09122, WO 97/03195, WO 96/21035, WO 91/07490, EP 0672 138, EP 0 270 618, EP 0 182 448, EP 0 162 067, EP 0 786 474, EP 0533 862, EP 0 506 757, EP 0 874 057, EP 0 795 021, EP 0 670 332, EP 0500 734, EP 0 232 112, EP 0 160 457, Sanberg et al., XXth Int. Congressof the World Fed. Of Hemophilia (1992), and Lind et al., Eur. J.Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII canbe obtained using recombinant methods or by deriving the sequence from avector known to include the same. Furthermore, the desired sequence canbe isolated directly from cells and tissues containing the same, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can alsobe produced synthetically, rather than cloned. The complete sequence canbe assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence [See, e.g., Edge,Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jayet al, J. Biol. Chem. 259:6311 (1984).

Furthermore, the invention is not limited to human Factor VIII. Indeed,it is intended that the present invention encompass Factor VIII fromanimals other than humans, including but not limited to companionanimals (e.g., canine, felines, and equines), livestock (e.g., bovines,caprines and ovines), laboratory animals, marine mammals, large cats,etc.

The AAV vectors may contain a nucleic acid coding for fragments ofFactor VIII which is itself not biologically active, yet whenadministered into the subject improves or restores the blood clottingtime. For example, as discussed above, the Factor VIII protein comprisestwo polypeptide chains: a heavy chain and a light chain separated by aB-domain which is cleaved during processing. As demonstrated by thepresent invention, co-tranducing recipient cells with the Factor VIIIheavy and light chains leads to the expression of biologically activeFactor VIII. Because, however, most hemophiliacs contain a mutation ordeletion in only one of the chain (e.g., heavy or light chain), it maybe possible to administer only the chain defective in the patient tosupply the other chain.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce “self”-directed antibodies. Tcell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors (TCRs) that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases.

C. Immunogenic Transgenes

Suitably, the AAV8 vectors of the invention avoid the generation ofimmune responses to the AAV8 sequences contained within the vector.However, these vectors may nonetheless be formulated in a manner whichpermits the expression of a transgene carried by the vectors to inducean immune response to a selected antigen. For example, in order topromote an immune response, the transgene may be expressed from aconstitutive promoter, the vector can be adjuvanted as described herein,and/or the vector can be put into degenerating tissue.

Examples of suitable immunogenic transgenes include those selected froma variety of viral families. Example of desirable viral families againstwhich an immune response would be desirable include, the picornavirusfamily, which includes the genera rhinoviruses, which are responsiblefor about 50% of cases of the common cold; the genera enteroviruses,which include polioviruses, coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus; and the genera apthoviruses,which are responsible for foot and mouth diseases, primarily innon-human animals. Within the picornavirus family of viruses, targetantigens include the VP1, VP2, VP3, VP4, and VPG. Other viral familiesinclude the astroviruses and the calcivirus family. The calcivirusfamily encompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatinencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis, and which include the putative cause of suddenacute respiratory syndrome (SARS). Within the coronavirus family, targetantigens include the E1 (also called M or matrix protein), E2 (alsocalled S or Spike protein), E3 (also called HE or hemagglutin-elterose)glycoprotein (not present in all coronaviruses), or N (nucleocapsid).Still other antigens may be targeted against the arterivirus family andthe rhabdovirus family. The rhabdovirus family includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus,parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. Another source of antigens is the bornavirus family.The reovirus family includes the genera reovirus, rotavirus (whichcauses acute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue). The retrovirus family includes the sub-family oncorivirinalwhich encompasses such human and veterinary diseases as feline leukemiavirus, HTLVI and HTLVII, lentivirinal (which includes HIV, simianimmunodeficiency virus, feline immunodeficiency virus, equine infectiousanemia virus, and spumavirinal). The papovavirus family includes thesub-family polyomaviruses (BKU and JCU viruses) and the sub-familypapillomavirus (associated with cancers or malignant progression ofpapilloma). The adenovirus family includes viruses (EX, AD7, ARD, O.B.)which cause respiratory disease and/or enteritis. The parvovirus familyfeline parvovirus (feline enteritis), feline panleucopeniavirus, canineparvovirus, and porcine parvovirus. The herpesvirus family includes thesub-family alphaherpesvirinae, which encompasses the genera simplexvirus(HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma), humanherpesviruses 6A, 6B and 7, Kaposi's sarcoma-associated herpesvirus andcercopithecine herpesvirus (B virus), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola major (Smallpox) and Vaccinia (Cowpox)),parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,and the sub-family entomopoxvirinae. The hepadnavirus family includesthe Hepatitis B virus. One unclassified virus which may be suitablesource of antigens is the Hepatitis delta virus, Hepatitis E virus, andprions. Another virus which is a source of antigens is Nipan Virus.Still other viral sources may include avian infectious bursal diseasevirus and porcine respiratory and reproductive syndrome virus. Thealphavirus family includes equine arteritis virus and variousEncephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci (and the toxinsproduced thereby, e.g., enterotoxin B); and streptococci. Pathogenicgram-negative cocci include meningococcus; gonococcus. Pathogenicenteric gram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucellaspecies (brucellosis); Francisella tularensis (which causes tularemia);Yersinia pestis (plague) and other yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism (Clostridum botulinum and its toxin); Clostridium perfringensand its epsilon toxin; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude glanders (Burkholderia mallei); actinomycosis; nocardiosis;cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;candidiasis, aspergillosis, and mucormycosis; sporotrichosis;paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma andchromomycosis; and dermatophytosis. Rickettsial infections includeTyphus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti),and Rickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis; trypanosomiasis;toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii;babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis;nematodes; trematodes or flukes; and cestode (tapeworm) infections.

Many of these organisms and/or the toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding CTLs to eliminate those T cells. In rheumatoid arthritis (RA),several specific variable regions of TCRs which are involved in thedisease have been characterized. These TCRs include V-3, V-14, V-17 andV-17. Thus, delivery of a nucleic acid sequence that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in RA. In multiple sclerosis (MS), severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and V-10. Thus, delivery of anucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28 and V-12. Thus,delivery of a nucleic acid molecule that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in scleroderma.

Thus, a rAAV8-derived recombinant viral vector of the invention providesan efficient gene transfer vehicle which can deliver a selectedtransgene to a selected host cell in vivo or ex vivo even where theorganism has neutralizing antibodies to one or more AAV serotypes. Inone embodiment, the rAAV and the cells are mixed ex vivo; the infectedcells are cultured using conventional methodologies; and the transducedcells are re-infused into the patient.

These compositions are particularly well suited to gene delivery fortherapeutic purposes and for immunization, including inducing protectiveimmunity. Further, the compositions of the invention may also be usedfor production of a desired gene product in vitro. For in vitroproduction, a desired product (e.g., a protein) may be obtained from adesired culture following transfection of host cells with a rAAVcontaining the molecule encoding the desired product and culturing thecell culture under conditions which permit expression. The expressedproduct may then be purified and isolated, as desired. Suitabletechniques for transfection, cell culturing, purification, and isolationare known to those of skill in the art.

The following examples illustrate several aspects and embodiments of theinvention.

Example 1 Production of Recombinant AAV8 Viral Genomes Equipped withAAV2 ITRs

Chimeric packaging constructs are generated by fusing AAV2 rep with capsequences of novel AAV serotypes. These chimeric packaging constructsare used, initially, for pseudotyping recombinant AAV genomes carryingAAV2 ITRs by triple transfection in 293 cell using Ad5 helper plasmid.These pseudotyped vectors are used to evaluate performance intransduction-based serological studies and evaluate gene transferefficiency of novel AAV serotypes in different animal models includingNHP and rodents, before intact and infectious viruses of these novelserotypes are isolated.

A. pAAV2GFP

The AAV2 plasmid which contains the AAV2 ITRs and green fluorescentprotein expressed under the control of a constitutive promoter. Thisplasmid contains the following elements: the AAV2 ITRs, a CMV promoterand the GFP coding sequences.

B. Cloning of Trans Plasmid

To construct the chimeric trans-plasmid for production of recombinantpseudotyped AAV8 vectors, p5E18 plasmid (Xiao et al., 1999, J. Virol73:3994-4003) was partially digested with Xho I to linearize the plasmidat the Xho I site at the position of 3169 bp only. The Xho I cut endswere then filled in and ligated back. This modified p5E18 plasmid wasrestricted with Xba I and Xho I in a complete digestion to remove theAAV2 cap gene sequence and replaced with a 2267 bp Spe I/Xho I fragmentcontaining the AAV8 cap gene which was isolated from pCRAAV8 6-5+15-4plasmid.

The resulting plasmid contains the AAV2 rep sequences for Rep78/68 underthe control of the AAV2 P5 promoter, and the AAV2 rep sequences forRep52/40 under the control of the AAV2 P19 promoter. The AAV9 capsidsequences are under the control of the AAV2 P40 promoter, which islocated within the Rep sequences. This plasmid further contains a spacer5′ of the rep ORF.

Alternatively, a similar plasmid can be constructed which utilizes theAAV8 rep sequences and the native AAV8 promoter sequences. This plasmidis then used for production of rAAV8, as described herein.

C. Production of Pseudotyped rAAV

The rAAV particles (AAV2 vector in AAV8 capsid) are generated using anadenovirus-free method. Briefly, the cis plasmid (pAAV2.1 lacZ plasmidcontaining AAV2 ITRs), and the trans plasmid pCRAAV8 6-5+15-4(containing the AAV2 rep and AAV8cap) and a helper plasmid,respectively, were simultaneously co-transfected into 293 cells in aratio of 1:1:2 by calcium phosphate precipitation.

For the construction of the pAd helper plasmids, pBG10 plasmid waspurchased from Microbix (Canada). A RsrII fragment containing L2 and L3was deleted from pBHG10, resulting in the first helper plasmid, pAdΔF13. Plasmid Ad F1 was constructed by cloning Asp700/SalI fragment witha PmeI/Sgfl deletion, isolating from pBHG10, into Bluescript. MLP, L2,L2 and L3 were deleted in the pAdΔF1. Further deletions of a 2.3 kb NruIfragment and, subsequently, a 0.5 kb RsrII/NruI fragment generatedhelper plasmids pAdΔF5 and pAdΔF6, respectively. The helper plasmid,termed pΔF6, provides the essential helper functions of E2a and E4 ORF6not provided by the E1-expressing helper cell, but is deleted ofadenoviral capsid proteins and functional E1 regions).

Typically, 50 μg of DNA (cis:trans:helper) was transfected onto a 150 mmtissue culture dish. The 293 cells were harvested 72 hourspost-transfection, sonicated and treated with 0.5% sodium deoxycholate(37° C. for 10 min.) Cell lysates were then subjected to two rounds of aCsCl gradient. Peak fractions containing rAAV vector are collected,pooled and dialyzed against PBS.

Example 2 Evaluation of Vectors with AAV8 Capsids

Vectors based on AAV1 (2/1), AAV5 (2/5) and AAV2 (2/2) were developedessentially as described for AAV8 in Example 1. Genome copy (GC) titersof AAV vectors were determined by TaqMan analysis using probes andprimers targeting SV40 poly A region as described previously [Gao, G.,et al., (2000) Hum Gene Ther 11, 2079-91]. Recombinant virions wererecovered by CsCl₂ sedimentation in all cases except AAV2/2, which waspurified by heparin chromatography.

Vectors were constructed for each serotype for a number of in vitro andin vivo studies. Eight different transgene cassettes were incorporatedinto the vectors and recombinant virions were produced for eachserotype. The recovery of virus, based on genome copies, is summarizedin Table 1. The yields of vector were high for each serotype with noconsistent differences between serotypes. Data presented in the tableare average genome copy yields with standard deviation×10¹³ of multipleproduction lots of 50 plate (150 mm) transfections.

TABLE 1 Production of Recombinant Vectors AAV2/1 AAV2/2 AAV2/5 AAV2/8CMV 7.30 ± 4.33 4.49 ± 2.89 5.19 ± 5.19 0.87 LacZ (n = 9) (n = 6) (n =8) (n = 1) CMV 6.43 ± 2.42 3.39 ± 2.42 5.55 ± 6.49 3.74 ± 3.88 EGFP (n =2) (n = 2) (n = 4) (n = 2) TBG 4.18  0.23 0.704 ± 0.43   0.532 LacZ (n= 1) (n = 1) (n = 2) (n = 1) Alb 4.67 ± 0.75 4.77 4.09 2.02 A1AT (n = 2)(n = 1) (n = 1) (n = 1) CB 0.567  0.438 2.82 0.816 ± 0.679 A1AT (n = 1)(n = 1) (n = 1) (n = 2) CMV 8.78 ± 2.37 1.43 ± 1.18 1.63 ± 1.15 1.32 ±0.87 rhCG (n = 7) (n = 2) (n = 3) (n = 3) TBG 8.51 ± 6.65 3.47 ± 2.095.26 ± 3.85 1.83 ± 0.98 rhCG (n = 6) (n = 5) (n = 4) (n = 5) TBG 1.24 ±1.29  0.63 ± 0.394 3.74 ± 2.48 15.8 ± 15.0 cFIX (n = 3) (n = 6) (n = 7)(n = 5)

Example 3 Serologic Analysis of Pseudotyped Vectors

C57BL/6 mice were injected with vectors of different serotypes ofAAVCBA1AT vectors intramuscularly (5×10¹¹ GC) and serum samples werecollected 34 days later. To test neutralizing and cross-neutralizingactivity of sera to each serotype of AAV, sera was analyzed in atransduction based neutralizing antibody assay [Gao, G. P., et al.,(1996) J Virol 70, 8934-43]. More specifically, the presence ofneutralizing antibodies was determined by assessing the ability of serumto inhibit transduction of 84-31 cells by reporter viruses (AAVCMVEGFP)of different serotypes. Specifically, the reporter virus AAVCMVEGFP ofeach serotype [at multiplicity of infection (MOI) that led to atransduction of 90% of indicator cells] was pre-incubated withheat-inactivated serum from animals that received different serotypes ofAAV or from naïve mice. After 1-hour incubation at 37° C., viruses wereadded to 84-31 cells in 96 well plates for 48 or 72-hour, depending onthe virus serotype. Expression of GFP was measured by FluoroImagin(Molecular Dynamics) and quantified by Image Quant Software.Neutralizing antibody titers were reported as the highest serum dilutionthat inhibited transduction to less than 50%.

The availability of GFP expressing vectors simplified the development ofan assay for neutralizing antibodies that was based on inhibition oftransduction in a permissive cell line (i.e., 293 cells stablyexpressing E4 from Ad5). Sera to selected AAV serotypes were generatedby intramuscular injection of the recombinant viruses. Neutralization ofAAV transduction by 1:20 and 1:80 dilutions of the antisera wasevaluated (Table 2). Antisera to AAV1, AAV2, AAV5 and AAV8 neutralizedtransduction of the serotype to which the antiserum was generated (AAV5and AAV8 to a lesser extent than AAV 1 and AAV2) but not to the otherserotype (i.e., there was no evidence of cross neutralization suggestingthat AAV 8 is a truly unique serotype).

TABLE 2 Serological Analysis of New AAV Serotypes. Serum dilution: Serumdilution: Serum dilution: Serum dilution: Sera: Immunization Vector 1/201/80 1/20 1/80 1/20 1/80 1/20 1/80 Group 1 AAV2/1 0 0 100 100 100 100100 100 Group 2 AAV2/2 100 100 0 0 100 100 100 100 Group 3 AAV2/5 100100 100 100 16.5 16.5 100 100 Group 4 AAV2/8 100 100 100 100 100 10026.3 60

Human sera from 52 normal subjects were screened for neutralizationagainst selected serotypes. No serum sample was found to neutralizeAAV2/8 while AAV2/2 and AAV2/1 vectors were neutralized in 20% and 10%of sera, respectively. A fraction of human pooled IqG representing acollection of 60,000 individual samples did not neutralize AAV2/8,whereas AAV2/2 and AAV2/1 vectors were neutralized at titers of serumequal to 1/1280 and 1/640, respectively.

Example 4 In Vivo Evaluation of Different Serotypes of AAV Vectors

In this study, 7 recombinant AAV genomes, AAV2CBhA1AT, AAV2A1bhA1AT,AAV2CMVrhCG, AAV2TBGrhCG, AAV2TBGcFIX, AAV2CMVLacZ and AAV2TBGLacZ werepackaged with capsid proteins of different serotypes. In all 7constructs, minigene cassettes were flanked with AAV2 ITRs. cDNAs ofhuman α-antitrypsin (A1AT) [Xiao, W., et al., (1999) J Virol 73,3994-4003] β-subunit of rhesus monkey choriogonadotropic hormone (CG)[Zoltick, P. W. & Wilson, J. M. (2000) Mol Ther 2, 657-9] canine factorIX [Wang, L., et al., (1997) Proc Natl Acad Sci USA 94, 11563-6] andbacterial β-glactosidase (i.e., Lac Z) genes were used as reportergenes. For liver-directed gene transfer, either mouse albumin genepromoter (Alb) [Xiao, W. (1999), cited above] or human thyroid hormonebinding globulin gene promoter (TBG) [Wang (1997), cited above] was usedto drive liver specific expression of reporter genes. In muscle-directedgene transfer experiments, either cytomegalovirus early promoter (CMV)or chicken β-actin promoter with CMV enhancer (CB) was employed todirect expression of reporters.

For muscle-directed gene transfer, vectors were injected into the righttibialis anterior of 4-6 week old NCR nude or C57BL/6 mice (Taconic,Germantown, N.Y.) at a dose of 1×10¹¹ genome copies (GC) per animal. Inliver-directed gene transfer studies, vectors were infused intraportallyinto 7-9 week old NCR nude or C57BL/6 mice (Taconic, Germantown, N.Y.),also at a dose of 1×10¹¹ genome copies (GC) per animal Serum sampleswere collected intraorbitally at different time points after vectoradministration. Muscle and liver tissues were harvested at differenttime points for cryosectioning and Xgal histochemical staining fromanimals that received the lacZ vectors. For the re-administrationexperiment, C56BL/6 mice initially received AAV2/1, 2/2, 2/5, 2/7 and2/8CBA1AT vectors intramuscularly and followed for A1AT gene expressionfor 7 weeks. Animals were then treated with AAV2/8TBGcFIX intraportallyand studied for cFIX gene expression.

ELISA based assays were performed to quantify serum levels of hA1AT,rhCG and cFIX proteins as described previously [Gao, G. P., et al.,(1996) J Virol 70, 8934-43; Zoltick, P. W. & Wilson, J. M. (2000) MolTher 2, 657-9; Wang, L., et al., Proc Natl Acad Sci USA 94, 11563-6].The experiments were completed when animals were sacrificed for harvestof muscle and liver tissues for DNA extraction and quantitative analysisof genome copies of vectors present in target tissues by TaqMan usingthe same set of primers and probe as in titration of vector preparations[Zhang, Y., et al., (2001) Mol Ther 3, 697-707].

The performance of vectors base on the new serotypes were evaluated inmurine models of muscle and liver-directed gene transfer and compared tovectors based on the known serotypes AAV1, AAV2 and AAV5. Vectorsexpressing secreted proteins (A1AT and CG-Table 3) were used toquantitate relative transduction efficiencies between differentserotypes through ELISA analysis of sera. The cellular distribution oftransduction within the target organ was evaluated using lacZ expressingvectors and X-gal histochemistry.

The performance of AAV vectors in skeletal muscle was analyzed followingdirect injection into the tibialis anterior muscles. Vectors containedthe same AAV2 based genome with the immediate early gene of CMV or a CMVenhanced β-actin promoter driving expression of the transgene. Previousstudies indicated that immune competent C57BL/6 mice elicit limitedhumoral responses to the human A1AT protein when expressed from AAVvectors [Xiao, W., et al., (1999) J Virol 73, 3994-4003].

In each strain, AAV2/1 vector produced the highest levels of A1AT andAAV2/2 vector the lowest, with AAV2/8 vectors showing intermediatelevels of expression. Peak levels of CG at 28 days following injectionof nu/nu NCR mice showed the highest levels from AAV2/7 and the lowestfrom AAV2/2 with AAV2/8 and AAV2/1 in between. Injection of AAV2/1 lacZvectors yielded gene expression at the injection sites in all musclefibers with substantially fewer lacZ positive fibers observed withAAV2/2 and AAV 2/8 vectors.

Similar murine models were used to evaluate liver-directed genetransfer. Identical doses of vector based on genome copies were infusedinto the portal veins of mice that were analyzed subsequently forexpression of the transgene. Each vector contained an AAV2 based genomeusing previously described liver-specific promoters (i.e., albumin orthyroid hormone binding globulin) to drive expression of the transgene.More particularly, CMVCG and TBGCG minigene cassettes were used formuscle and liver-directed gene transfer, respectively. Levels of rhCGwere defined as relative units (rUs×10³). The data were from assayingserum samples collected at day 28, post vector administration (4 animalsper group). As shown in Table 4, the impact of capsid proteins on theefficiency of transduction of A1AT vectors in nu/nu and C57BL/6 mice andCG vectors in C57BL/6 mice was consistent, i.e., AAV2/8 is the mostefficient for pseudotype for liver-directed gene transfer.

TABLE 3 Expression of β-unit of Rhesus Monkey Chorionic Gonadotropin(rhCG) in Mouse Muscle and Liver. Vector Muscle Liver AAV2/1 4.5 ± 2.11.6 ± 1.0 AAV2 0.5 ± 0.1 0.7 ± 0.3 AAV2/5 ND* 4.8 ± 0.8 AAV2/8 4.0 ± 0.776.0 ± 22.8 *Not determined in this experiment.

In all cases, AAV2/8 vectors yielded the highest levels of transgeneexpression that ranged from 16 to 110 greater than what was obtainedwith AAV2/2 vectors; expression from AAV2/5 was intermediate. Analysisof X-Gal stained liver sections of animals that received thecorresponding lacZ vectors showed a correlation between the number oftransduced cells and overall levels of transgene expression. DNAsextracted from livers of C57BL/6 mice who received the A1AT vectors wereanalyzed for abundance of vector DNA using real time PCR technology.

The amount of vector DNA found in liver 56 days after injectioncorrelated with the levels of transgene expression (Table 4). For thisexperiment, a set of probe and primers targeting the SV40 polyA regionof the vector genome was used for TaqMan PCR. Values shown are means ofthree individual animals with standard deviations. The animals weresacrificed at day 56 to harvest liver tissues for DNA extraction. Thesestudies indicate that AAV8 is the most efficient vector forliver-directed gene transfer due to increased numbers of transducedhepatocytes.

TABLE 4 Real Time PCR Analysis for Abundance of AAV Vectors in nu/nuMouse Liver Following Injection of 1 × 10¹¹ Genome Copies of Vector. AAVvectors/Dose Genome Copies per Cell AAV2/1AlbA1AT  0.6 ± 0.36AAV2AlbA1AT 0.003 ± 0.001 AAV2/5AlbA1AT 0.83 ± 0.64 AAV2/8AlbA1AT 18 ±11

The serologic data described above suggest that AAV2/8 vector should notbe neutralized in vivo following immunization with the other serotypes.C57BL/6 mice received intraportal injections of AAV2/8 vector expressingcanine factor IX (10¹¹ genome copies) 56 days after they receivedintramuscular injections of A1AT vectors of different serotypes. Highlevels of factor IX expression were obtained 14 days following infusionof AAV2/8 into naïve animals (17±2 μg/ml, N=4) which were notsignificantly different that what was observed in animals immunized withAAV2/1 (31±23 μg/ml, N=4), and AAV2/2 (16 μg/ml, N=2). This contrasts towhat was observed in AAV2/8 immunized animals that were infused with theAAV2/8 factor IX vector in which no detectable factor IX was observed(<0.1 μg/ml, N=4).

Oligonucleotides to conserved regions of the cap gene did amplifysequences from rhesus monkeys that represented unique AAVs. Identicalcap signature sequences were found in multiple tissues from rhesusmonkeys derived from at least two different colonies. Full-length repand cap open reading frames were isolated and sequenced from singlesources. Only the cap open reading frames of the novel AAVs werenecessary to evaluate their potential as vectors because vectors withthe AAV8 capsids were generated using the ITRs and rep from AAV2. Thisalso simplified the comparison of different vectors since the actualvector genome is identical between different vector serotypes. In fact,the yields of recombinant vectors generated using this approach did notdiffer between serotypes.

Vectors based on AAV8 appear to be immunologically distinct (i.e., theyare not neutralized by antibodies generated against other serotypes).Furthermore, sera from humans do not neutralize transduction by AAV8vectors, which is a substantial advantage over the human derived AAVscurrently under development for which a significant proportion of thehuman population has pre-existing immunity that is neutralizing[Chirmule, N., et al., (1999) Gene Ther 6, 1574-83].

The tropism of the new vector is favorable for in vivo applicationsImportantly, AAV2/8 provides a substantial advantage over the otherserotypes in terms of efficiency of gene transfer to liver that untilnow has been relatively disappointing in terms of the numbers ofhepatocytes stably transduced. AAV2/8 consistently achieved a 10 to100-fold improvement in gene transfer efficiency as compared to theother vectors. The basis for the improved efficiency of AAV2/8 isunclear, although it presumably is due to uptake via a differentreceptor that is more active on the basolateral surface of hepatocytes.This improved efficiency will be quite useful in the development ofliver-directed gene transfer where the number of transduced cells iscritical, such as in urea cycle disorders and familialhypercholesterolemia.

Thus, the lack of pre-existing immunity to AAV8 and the favorabletropism of the vectors for liver indicates that vectors with AAV8 capsidproteins are suitable for use as vectors in human gene therapy and otherin vivo applications.

Example 5 Tissue Tropism Studies

In the design of a high throughput functional screening scheme for novelAAV constructs, a non-tissue specific and highly active promoter, CBpromoter (CMV enhanced chicken β-actin promoter) was selected to drivean easily detectable and quantifiable reporter gene, humanα-anti-trypsin gene. Thus only one vector for each new AAV clone needsto be made for gene transfer studies targeting 3 different tissues,liver, lung and muscle to screen for tissue tropism of a particular AAVconstruct. The following table summarizes data generated from novel AAVvectors in the tissue tropism studies (AAVCBA1AT). Table 5 reports dataobtained (in μg A1AT/mL serum) at day 14 of the study.

TABLE 5 Target Tissue Vector Lung Liver Muscle AAV2/1 ND ND 45 ± 11AAV2/5 0.6 ± 0.2 ND ND AAV2/8 ND 84 ± 30 ND

AAV vector carried CC10hA1AT minigene for lung specific expression werepseudotyped with capsids of novel AAVs were given to Immune deficientanimals (NCR nude) in equal volume (50 μl each of the original prepswithout dilution) via intratracheal injections as provided in thefollowing table. The vectors were also administered to immune competentanimals (C57BL/6) in equal genome copies (1×10¹¹ GC) as shown in theTable 6. (1×10¹¹ GC per animal, C57BL/6, day 14, detection limit ≧0.033μg/ml). As shown, AAV8 is the best liver transducer.

TABLE 6 AAV μg of A1AT/ml Vector with 1 × 10¹¹ vector 2/1 0.076 ± 0.0312/2  0.1 ± 0.09 2/5 0.0840.033 2/8 1.92 ± 1.3 

Example 6 Model of Hypercholesterolemia

To further assess the effect of rAAV-mediated transgene expression bythe AAV2/8 constructs of the invention, a further study was performed.

A. Vector Construction

AAV vectors packaged with AAV8 capsid proteins were constructed using apseudotyping strategy [Hildinger M, et al., J. Virol 2001;75:6199-6203]. Recombinant AAV genomes with AAV2 inverted terminalrepeats (ITR) were packaged by triple transfection of 293 cells with thecis-plasmid, the adenovirus helper plasmid and a chimeric packagingconstruct, a fusion of the capsids of the novel AAV serotypes with therep gene of AAV2. The chimeric packaging plasmid was constructed aspreviously described [Hildinger et al, cited above]. The recombinantvectors were purified by the standard CsCl₂ sedimentation method. Todetermine the yield TaqMan (Applied Biosystems) analysis was performedusing probes and primers targeting the SV40 poly(A) region of thevectors [Gao G P, et al., Hum Gene Ther. 2000 Oct. 10; 11(15):2079-91].The resulting vectors express the transgene under the control of thehuman thyroid hormone binding globulin gene promoter (TBG).

B. Animals

LDL receptor deficient mice on the C57Bl/6 background were purchasedfrom the Jackson Laboratory (Bar Harbor, Me., USA) and maintained as abreeding colony. Mice were given unrestricted access to water andobtained a high fat Western Diet (high % cholesterol) starting threeweeks prior vector injection. At day −7 as well at day 0, blood wasobtained via retroorbital bleeds and the lipid profile evaluated. Themice were randomly divided into seven groups. The vector was injectedvia an intraportal injection as previously described ([Chen S J et al.,Mol Therapy 2000; 2(3), 256-261]. Briefly, the mice were anaesthetizedwith ketamine and xylazine. A laparotomy was performed and the portalvein exposed. Using a 30 g needle the appropriate dose of vector dilutedin 100 μl PBS was directly injected into the portal vein. Pressure wasapplied to the injection site to ensure a stop of the bleeding. The skinwound was closed and draped and the mice carefully monitored for thefollowing day. Weekly bleeds were performed starting at day 14 afterliver directed gene transfer to measure blood lipids. Two animals ofeach group were sacrificed at the timepoints week 6 and week 12 aftervector injection to examine atherosclerotic plaque size as well asreceptor expression. The remaining mice were sacrificed at week 20 forplaque measurement and determination of transgene expression.

TABLE 7 Vector dose N Group 1 AAV2/8-TBG-hLDLr 1 × 10¹² gc 12 Group 2AAV2/8-TBG-hLDLr 3 × 10¹¹ gc 12 Group 3 AAV2/8-TBG-hLDLr 1 × 10¹¹ gc 12

C. Serum Lipoprotein and Liver Function Analysis

Blood samples were obtained from the retroorbital plexus after a 6 hourfasting period. The serum was separated from the plasma bycentrifugation. The amount of plasma lipoproteins and livertransaminases in the serum were detected using an automatized clinicalchemistry analyzer (ACE, Schiapparelli Biosystems, Alpha Wassermann)

D. Detection of Transgene Expression

LDL receptor expression was evaluated by immuno-fluorescence stainingand Western blotting. For Western Blot frozen liver tissue washomogenized with lysis buffer (20 mM Tris, pH 7.4, 130 mM NaCl, 1%Triton X 100, proteinase inhibitor complete, EDTA-free, Roche, Mannheim,Germany) Protein concentration was determined using the Micro BCAProtein Assay Reagent Kit (Pierce, Rockford, Ill.). 40 μg of protein wasresolved on 4-15% Tris-HCl Ready Gels (Biorad, Hercules, Calif.) andtransferred to a nitrocellulose membrane (Invitrogen). To generateanti-hLDL receptor antibodies a rabbit was injected intravenously withan AdhLDLr prep (1×10¹³ gc). Four weeks later the rabbit serum wasobtained and used for Western Blot. A 1:100 dilution of the serum wasused as a primary antibody followed by a HRP-conjugated anti-rabbit IgGand ECL chemiluminescent detection (ECL Western Blot Detection Kit,Amersham, Arlington Heights, Ill.).

D. Immunocytochemistry

For determination of LDL receptor expression in frozen liver sectionsimmunohistochemistry analyses were performed. 10 um cryostat sectionswere either fixed in acetone for 5 minutes, or unfixed. Blocking wasobtained via a 1 hour incubation period with 10% of goat serum. Sectionswere then incubated for one hour with the primary antibody at roomtemperature. A rabbit polyclonal antibody anti-human LDL (BiomedicalTechnologies Inc., Stoughton, Mass.) was used diluted accordingly to theinstructions of the manufacturer. The sections were washed with PBS, andincubated with 1:100 diluted fluorescein goat anti-rabbit IgG (Sigma, StLouis, Mo.). Specimens were finally examined under fluorescencemicroscope Nikon Microphot-FXA. In all cases, each incubation wasfollowed by extensive washing with PBS. Negative controls consisted ofpreincubation with PBS, omission of the primary antibody, andsubstitution of the primary antibody by an isotype-matched non-immunecontrol antibody. The three types of controls mentioned above wereperformed for each experiment on the same day.

E. Gene Transfer Efficiency

Liver tissue was obtained after sacrificing the mice at the designatedtime points. The tissue was shock frozen in liquid nitrogen and storedat −80° C. until further processing. DNA was extracted from the livertissue using a QIAamp DNA Mini Kit (QIAGEN GmbH, Germany) according tothe manufacturers protocol. Genome copies of AAV vectors in the livertissue were evaluated using Taqman analysis using probes and primersagainst the SV40 poly(A) tail as described above.

F. Atherosclerotic Plaque Measurement

For the quantification of the atherosclerotic plaques in the mouse aortathe mice were anaesthetized (10% ketamine and xylazine, ip), the chestopened and the arterial system perfused with ice-cold phosphate bufferedsaline through the left ventricle. The aorta was then carefullyharvested, slit down along the ventral midline from the aortic arch downto the femoral arteries and fixed in formalin. The lipid-richatherosclerotic plaques were stained with Sudan IV (Sigma, Germany) andthe aorta pinned out flat on a black wax surface. The image was capturedwith a Sony DXC-960 MD color video camera. The area of the plaque aswell as of the complete aortic surface was determined using Phase 3Imaging Systems (Media Cybernetics).

G. Clearance of I¹²⁵ LDL

Two animals per experimental group were tested. A bolus of I¹²⁵-labeledLDL (generously provided by Dan Rader, Upenn) was infused slowly throughthe tail vein over a period of 30 sec (1,000,000 counts of [I¹²⁵]-LDLdiluted in 100 μl sterile PBS/animal). At time points 3 min, 30 min, 1.5hr, 3 hr, 6 hr after injection a blood sample was obtained via theretro-orbital plexus. The plasma was separated off from the whole bloodand 10 μl plasma counted in the gamma counter. Finally the fractionalcatabolic rate was calculated from the lipoprotein clearance data.

H. Evaluation of Liver Lipid Accumulation

Oil Red Staining of frozen liver sections was performed to determinelipid accumulation. The frozen liver sections were briefly rinsed indistilled water followed by a 2 minute incubation in absolute propyleneglycol. The sections were then stained in oil red solution (0.5% inpropylene glycol) for 16 hours followed by counterstaining with Mayer'shematoxylin solution for 30 seconds and mounting in warmed glycerinjelly solution.

For quantification of the liver cholesterol and triglyceride contentliver sections were homogenized and incubated in chloroform/methanol(2:1) overnight. After adding of 0.05% H₂SO₄ and centrifugation for 10minutes, the lower layer of each sample was collected, divided in twoaliquots and dried under nitrogen. For the cholesterol measurement thedried lipids of the first aliquot were dissolved in 1% Triton X-100 inchloroform. Once dissolved, the solution was dried under nitrogen. Afterdissolving the lipids in ddH₂0 and incubation for 30 minutes at 37° C.the total cholesterol concentration was measured using a TotalCholesterol Kit (Wako Diagnostics). For the second aliquot the driedlipids were dissolved in alcoholic KOH and incubated at 60° C. for 30minutes. Then 1M MgCl₂ was added, followed by incubation on ice for 10minutes and centrifugation at 14,000 rpm for 30 minutes. The supernatantwas finally evaluated for triglycerides (Wako Diagnostics).

All of the vectors pseudotyped in an AAV2/8 capsid lowered totalcholesterol, LDL and triglycerides as compared to the control. Thesetest vectors also corrected phenotype of hypercholesterolemia in adose-dependent manner. A reduction in plaque area for the AAV2/8 micewas observed in treated mice at the first test (2 months), and theeffect was observed to persist over the length of the experiment (6months).

Example 7 Functional Factor IX Expression and Correction of Hemophilia

A. Knock-Out Mice

Functional canine factor IX (FIX) expression was assessed in hemophiliaB mice. Vectors with capsids of AAV1, AAV2, AAV5 or AAV8 wereconstructed to deliver AAV2 5′ ITR—liver-specific promoter [LSP]—canineFIX—woodchuck hepatitis post-regulatory element (WPRE)—AAV2 3′ ITR. Thevectors were constructed as described in Wang et al, 2000, MolecularTherapy 2: 154-158), using the appropriate capsids.

Knock-out mice were generated as described in Wang et al, 1997. Proc.Natl. Acad. Sci. USA 94: 11563-11566. This model closely mimic thephenotypes of hemophilia B in human.

Vectors of different serotypes were delivered as a single intraportalinjection into the liver of adult hemophiliac C57Bl/6 mice in a dose of1×10¹¹ GC/mouse for the five different serotypes and a second AAV8vector was also delivered at 1×10¹⁰ GC/mouse. Control group was injectedwith 1×10¹¹ GC of AAV2/8 TBG LacZ3. Each group contains 5-10 male andfemale mice. Mice were bled bi-weekly after vector administration.

1. ELISA

The canine FIX concentration in the mouse plasma was determined by anELISA assay specific for canine factor IX, performed essentially asdescribed by Axelrod et al, 1990, Proc. Natl. Acad. Sci. USA,87:5173-5177 with modifications. Sheep anti-canine factor IX (EnzymeResearch Laboratories) was used as primary antibody and rabbitanti-canine factor IX ((Enzyme Research Laboratories) was used assecondary antibody. Beginning at two weeks following injection,increased plasma levels of cFIX were detected for all test vectors. Theincreased levels were sustained at therapeutic levels throughout thelength of the experiment, i.e., to 12 weeks. Therapeutic levels areconsidered to be 5% of normal levels, i.e., at about 250 ng/mL.

The highest levels of expression were observed for the AAV2/8 (at 10¹¹),with sustained superphysiology levels cFIX levels (ten-fold higher thanthe normal level). Expression levels for AAV2/8 (10¹¹) wereapproximately 10 fold higher than those observed for AAV2/2 and AAV2/8(10¹⁰). The lowest expression levels, although still above thetherapeutic range, were observed for AAV2/5.

2. In Vitro Activated Partial Thromboplastin Time (aPTT) Assay

Functional factor IX activity in plasma of the FIX knock-out mice wasdetermined by an in vitro activated partial thromboplastin time (aPTT)assay—Mouse blood samples were collected from the retro-orbital plexusinto 1/10 volume of citrate buffer. APTT assay was performed asdescribed by Wang et al, 1997, Proc. Natl. Acad. Sci. USA 94:11563-11566.

Clotting times by aPTT on plasma samples of all vector injected micewere within the normal range (approximately 60 sec) when measured at twoweeks post-injection, and sustained clotting times in the normal orshorter than normal range throughout the study period (12 weeks).

Lowest sustained clotting times were observed in the animals receivingAAV2/8 (10¹¹). By week 12, AAV2/2 also induced clotting times similar tothose for AAV2/8. However, this lowered clotting time was not observedfor AAV2/2 until week 12, whereas lowered clotting times (in the 25-40sec range) were observed for AAV2/8 beginning at week two.

Immuno-histochemistry staining on the liver tissues harvested from someof the treated mice is currently being performed. About 70-80% ofhepatocytes are stained positive for canine FIX in the mouse injectedwith AAV2/8.cFIX vector.

B. Hemophilia B Dogs

Dogs that have a point mutation in the catalytic domain of the F.IXgene, which, based on modeling studies, appears to render the proteinunstable, suffer from hemophilia B [Evans et al, 1989, Proc. Natl. Acad.Sci. USA, 86:10095-10099). A colony of such dogs has been maintained formore than two decades at the University of North Carolina, Chapel Hill.The homeostatic parameters of these doges are well described and includethe absence of plasma F.IX antigen, whole blood clotting times in excessof 60 minutes, whereas normal dogs are 6-8 minutes, and prolongedactivated partial thromboplastin time of 50-80 seconds, whereas normaldogs are 13-28 seconds. These dogs experience recurrent spontaneoushemorrhages. Typically, significant bleeding episodes are successfullymanaged by the single intravenous infusion of 10 ml/kg of normal canineplasma; occasionally, repeat infusions are required to control bleeding.

Four dogs were injected intraportally with AAV.cFIX according to theschedule below. A first dog received a single injection with AAV2/2.cFIXat a dose of 3.7×10¹¹ genome copies (GC)/kg and was sacrificed at day665 due to severe spinal hemorrhage. A second dog received a firstinjection of AAV2/2.cFIX (2.8×10¹¹ GC/kg), followed by a secondinjection with AAV2/5.cFIX (2.3×10¹³ GC/kg) at day 1180. A third dogreceived a single injection with AAV2/2.cFIX at a dose of 4.6×10¹²GC/kg. The fourth dog received an injection with AAV2/2.cFIX (2.8×10¹²GC/kg) and an injection at day 995 with AAV2/8.cFIX (5×10¹² GC/kg).

The abdomen of hemophilia dogs were aseptically and surgically openedunder general anesthesia and a single infusion of vector wasadministered into the portal vein. The animals were protected fromhemorrhage in the pen-operative period by intravenous administration ofnormal canine plasma. The dog was sedated, intubated to induce generalanesthesia, and the abdomen was shaved and prepped. After the abdomenwas opened, the spleen was moved into the operative field. The splenicvein was located and a suture was loosely placed proximal to a smalldistal incision in the vein. An introduced was rapidly inserted into thevein, then the suture loosened and a 5 F cannula was threaded to anintravenous location near the portal vein threaded to an intravenouslocation near the portal vein bifurcation. After hemostasis was securedand the catheter balloon was inflated, approximately 5.0 ml of vectordiluted in PBS was infused into the portal vein over a 5 minuteinterval. The vector infusion was followed by a 5.0 ml infusion ofsaline. The balloon was then deflated, the callula was removed andvenous hemostatis was secured. The spleen was then replaced, bleedingvessels were cauterized and the operative wound was closed. The animalwas extubated having tolerated the surgical procedure well. Bloodsamples were analyzed as described. [Wang et al, 2000, Molecular Therapy2: 154-158]

The results are summarized in the table below. Dog C51, female, was 13.6kg and 6.5 months old at the time of first injection. Dog C52, male, was17.6 kg and 6.5 months old at first injection; and 17.2 kg and 45.2months at second injection. Dog C55, male, was a 19.0 kg and 12.0 monthsat first injection. Dog D39, female, was a 5.0 kg and 2.8 months atfirst injection; 22.6 kg and 35.4 months old at the time of the secondinjection. In the table, GC refers to genome copies of the AAV vectors.WBCT were >60 minutes (except C52=42 min) before injection. BaselineaPTT for C51=98.4 sec, C52=97.7 sec; C55=145.1 sec; D39=97.8 sec. Bleedspost-treatment were spontaneous bleeding episodes happening inhemophilia B dogs post-AAV vector treatment that required treatment withplasma infusion.

TABLE 8 Hemophilia B Dogs Injected with rAAV intraportally Vector TotalAvg Avg Avg cFIX Dose GC WBCT aPTT plasma Dog Vector (GC/kg) Inject(min) (min) (ng/mL) 1^(st) C51 AAV2- 3.7 × 5 × 13.2 ± 77.5 ± 3.8 ±injec- LSP.cFIX 10¹¹ 10¹² 2.1 15.1 1.0 tion C52 AAV2- 2.8 × 5.0 × 16.1 ±81.5 ± 3.7 ± LSP.cFIX 10¹¹ 10¹² 3.5 17.7 1.1 C55 AAV2- 4.6 × 8.7 × 10.2± 46.4 ± 259.7 ± LSP.cFIX 10¹² 10¹³ 2.2 6.1 28.5 WPRE D39 AAV2- 2.8 ×1.4 × 11.5 ± 59.1 ± 34.4 ± LSPcFIX 10¹² 10¹³ 2.6 6.3 9.8 WPRE 2^(nd) C52AAV2/5- 2.3 × 4.0 × 12.9 ± 41.9 ± 817.3 ± injec- LSP.cFIX 10¹³ 10¹⁴ 1.12.7 102.1 tion WPRE D39 AAV2/8- 5.0 × 1.1 × 12.6 ± 656.9 ± LSP.cFIX 10¹²10¹⁴ 1.5 1.1 WPRE

1. Whole Blood Clotting Time (WBCT)

WBCT following injection with the AAV2/2 vectors were somewhat variable,ranging from about 6.5 min to 30 minutes. WBCT for a normal dog is 6-12min Sharp drops in WBCT were observed immediately upon injection withthe AAV2/8 or AAV2/5 vectors The sharp drop was also observed in C55injected with AAV2 (d2=9 min), and for C51 and C52, the early data pointfor WBCT were not checked. The sharp drop is believed to be due to thedog plasma infusion before and after the surgery. WBCT is an assay verysensitive to low level of FIX, it is not very sensitive to the actuallevel of FIX (aPTT is more relevant).

2. aPTT Assay

Clotting times by aPTT on plasma samples of all vector injected dogswere variable over the first approximately 700 days, at which timeclotting times leveled in the normal range (40-60 sec, normal dog: 24-32sec). A sharp drop into the normal range was observed following each ofthe second injections (AAV2/8 or AAV2/5). While clotting times were notsustained in the normal range, clotting times were reduced to levelsbelow those observed prior to the second injection.

For aPTT, normal dogs are 24-32 sec, and hemophilia B dogs are 80-106sec. For C51 and C52 who received low dose of AAV2.cFIX vector, averageaPTT after treatment remain at 77.5 and 81.5 sec, not significantlydifferent from hemophilia B dogs without treatment. Higher dose of AAV2improved the average aPTT to 59.1 and 46.4 sec, respectively for D39 andC55. After the treatment of AAV2/5, the average aPTT for C52 improvedsignificantly from 81.5 sec to 41.9 sec. And for D39, after the AAV2/8treatment, the average aPTT improve from 59.1 sec.

3. Canine Factor IX ELISA

cFIX levels were detectable following the first set of injections,albeit below therapeutic levels. Following injection with AAV2/8 andAAV2/5, levels of cFIX rose spiked into the therapeutic range and thenleveled off within the therapeutic range (normal is 5 μg/ml in plasma,therapeutic level is 5% of normal level which is 250 ng/ml).

The first three weeks of WBCT, aPTT and cFIX antigen are affected by thedog plasma infusion before and after the surgery. It is hard to concludethe drop of clotting time or the rise of cFIX antigen level is due tothe vector or the plasma infusion for the first 3 weeks. However, it isinteresting to note that the quick and dramatic rise of cFIX antigenafter 2/5 and 2/8 vector injection. This is unique to AAV2/5 and 2/8injected dogs and could be attributed to AAV2/5 and 2/8 vectors ratherthan the normal dog plasma infusion, since all dogs received similaramount of normal dog plasma infusion for the surgery. Three days afterAAV2/8 injection, the level of cFIX in the plasma of D39 reached 9.5μg/ml and peaked at 10.4 μg/ml at day 6, twice as much as the normallevel (5 μg/ml). The cFIX level gradually decreased to the average of817 ng/ml (C52, AAV2/5) and 657 ng/ml (D39, AAV2/8). In C52, 3 daysafter injection of AAV2/5 vector, the cFIX level reached 2.6 μg/ml andpeaked at 4.6 μg/ml at day 7. In C55, who received AAV2 vector at thedose similar to that of AAV2/8 injected to D39, peaked only at 2.2 μg/mlat day 3, then gradually dropped and maintained at 5% of normal level ofcFIX.

The doses of vector received by C55 (AAV2, 4.6×10¹² GC/kg) and thesecond injection in D39 (AAV2/8, 5×10¹² GC/kg) were very close. However,the cFIX expression levels raised in D39 by AAV2/8 vector (average657−34=623 ng/ml, 12.5% of normal level) was 2.5 fold higher than thatin C55 (average 259 ng/ml, 5% of normal level). This suggests AAV2/8 is2.5 fold more potent than AAV2 in dogs injected intraportally withsimilar dose of vectors. And in the same dog D39, the second injectionof two fold higher dose of AAV2/8 dramatically increased the cFIX levelfrom 0.7% to 13.1%, 18.7 fold higher than the first injection. And inC52, the second injection of 2.3×10¹³ GC/ml of AAV2/5 vector resulted inaverage 817 ng/ml (16.3% of normal level) of cFIX in the plasma. Thiswas only marginally higher (1.3 fold) than the cFIX level raised in D39by AAV2/8 (average 623 ng/ml, 12.5% of normal level). However, the doseof AAV2/5 injected in C52 was 4.6 fold higher than the dose of AAV2/8injected in D39. This suggests that AAV2/8 vector is also more potentthan AAV2/5 vector in dogs.

The first injection of AAV2 vectors did not block the success oftransduction by AAV2/5 and AAV2/8 vectors after the second injection indogs. Readministration using a different serotype of AAV vector can beused as an approach to treat animals or humans who have been previouslyexposed to AAV2 or treated with AAV2 vectors.

Example 8 Mouse Model of Liver Enzyme Disorder

The AAV2/8 vector generated as described herein was studied for itsefficiency in transferring the liver enzyme gene ornithinetranscarbamylase (OTC) in an accepted animal model for OTC deficiency[X. Ye et al, Pediatric Research, 41(4):527-534 (1997); X. Ye et al, J.Biol. Chem., 271(7):3639-3646 (February 1996)]. The results of thisexperiment (data not shown) demonstrate that an AAV2/8 vector of theinvention carrying the ornithine transcarbamylase (OTC) gene wasobserved to correct OTC deficiency.

Example 9 In Vivo Expression of Factor VIII

Three groups of C57BL/6 mice are injected via the portal vein witheither 3×10¹¹ genome copies AAV vector carrying the Factor VIII heavychain (FVIII-HC), 3×10¹¹ genome copies of AAV vector carrying FactorVIII light chain (FVIII-LC), or 3×10¹¹ particles of both AAV-FVIII-HCand AAV-FVIII-LC. In addition, a group of four animals is injected with3×10¹¹ particles of AAV carrying Factor IX (FIX), which is known to beuseful in treatment of hemophilia B. It has been shown that this strainof mice does not elicit an immune response to human FVIII when the geneis delivered to the liver via an adenoviral vector (Connelly et al.,Blood 87:4671-4677 [1996]).

These experiments will demonstrate the feasibility of producingbiologically active FVIII using two AAV vectors to independently deliverthe heavy and light chains of FVIII.

Blood samples are collected in sodium citrate via the retro-orbitalplexus at biweekly intervals for the first 2 months and at monthlyintervals thereafter for 6 months and at 11 months. Very high levels ofFVIII light chain will be expressed in animals injected withAAV-FVIII-LC alone or both vectors.

In order to assess the amount of biologically active human FVIIIproduced in the animals, a modified ChromZ assay is used. Since thisassay detects both human and murine FVIII, the amount of FVIII presentin the plasma before and after adsorption to an antibody specific tohuman FVIII is determined. The amount of FVIII remaining in the plasmaafter adsorption represents the amount of active murine FVIII and thedifference represented the amount of active human FVIII. The modifiedChromZ assay will indicate that only those animals injected with bothvectors produced biologically active FVIII.

The animals are expected to maintain physiological levels of activeprotein for more than 11 months, without waning.

All publications cited in this specification, and any sequence listingsassociated therewith, are incorporated herein by reference. While theinvention has been described with reference to particularly preferredembodiments, it will be appreciated that modifications can be madewithout departing from the spirit of the invention.

1: An adeno-associated virus (AAV)8 viral vector comprising an AAV8 capsid having packaged therein a heterologous gene operably linked to regulatory sequences which direct its expression, wherein the heterologous gene encodes factor IX, wherein the AAV8 capsid comprises a vp3 capsid protein having the sequence of aa 204 to 738 of SEQ ID NO: 2, or a sequence which is at least 95% identical to said sequence of aa 204 to 738 of SEQ ID NO:
 2. 2: The vector according to claim 1, further comprising one or more AAV inverted terminal repeat (ITR) sequence from an AAV heterologous to AAV8. 3: The vector according to claim 2, wherein the one or more AAV ITR is from AAV2. 4: The vector according to claim 1, wherein said vp3 capsid protein has a sequence which is at least 95% identical to the sequence of aa 204 to 738 of SEQ ID NO:
 2. 5: The vector according to claim 4, wherein said vp3 capsid protein has a sequence at least 99% identical to the sequence of aa 204 to 738 of SEQ ID NO:
 2. 6: The vector according to claim 5, wherein said vp3 capsid protein has the sequence of aa 204 to 738 of SEQ ID NO:
 2. 7: The vector according to claim 1, wherein said AAV8 capsid further comprises a vp1 capsid protein having a sequence which is at least 95% identical to the sequence of aa 1 to 738 of SEQ ID NO:
 2. 8: The vector according to claim 7, wherein said vp1 capsid protein has an amino acid sequence at least 99% identical to the sequence of aa 1 to 738 of SEQ ID NO:
 2. 9: The vector according to claim 8, wherein said vp1 capsid protein has the sequence of aa 1 to 738 of SEQ ID NO:
 2. 10: The vector according to claim 1, wherein said AAV8 capsid further comprises a vp2 capsid protein having a sequence which is at least 95% identical to the sequence of aa 138 to 738 of SEQ ID NO:
 2. 11: The vector according to claim 10, wherein said vp2 capsid protein has an amino acid sequence at least 99% identical to the sequence of aa 138 to 738 of SEQ ID NO:
 2. 12: The vector according to claim 11, wherein said vp2 capsid protein has the sequence of aa 138 to 738 of SEQ ID NO:
 2. 13: A host cell containing the vector according to claim 1 in culture. 14: A method of delivering a heterologous gene encoding factor IX to a cell, said method comprising contacting said cell with the vector according to claim
 1. 15: The method according to claim 14, comprising delivering said vector to a hepatocyte. 16: A method for treating hemophilia B, said method comprising the step of contacting a cell with a vector according to claim 1, wherein said vector directs expression of factor IX. 17: A composition comprising at least the vector according to claim 1 and a pharmaceutically acceptable carrier. 18: A composition comprising at least the vector according to claim 1 and a preservative or chemical stabilizer. 19: A composition comprising at least an adeno-associated virus (AAV) vector and a physiologically compatible carrier, the AAV vector comprising an AAV capsid comprising at least an AAV8 vp3 capsid protein having a sequence comprising amino acids 204 to 738 of SEQ ID NO: 2 or an amino acid sequence at least 95% identical thereto, said capsid having packaged therein a heterologous gene operably linked to regulatory sequences which direct its expression, wherein the heterologous gene encodes factor IX. 20: A composition comprising at least an adeno-associated virus (AAV) vector and a physiologically compatible carrier, the AAV vector comprising an AAV capsid comprising at least an AAV8 vp1 capsid protein having a sequence comprising amino acids 1 to 738 of SEQ ID NO: 2 or an amino acid sequence at least 95% identical thereto, said capsid having packaged therein a heterologous gene operably linked to regulatory sequences which direct its expression, wherein the heterologous gene encodes factor IX. 